Flow cytometry in combination with selective cell sorting (FACS: Fluorescence Activated Cell Sorting) is a method which became established more than 20 years ago. It allows the rapid screening and sorting of a large number of cells (sorting rates of up to 2000 cells/s). However, it has been predominantly used in the analytical field (medical diagnostics). Becton Dickinson, Beckman Coulter or Cytomation offer appropriate technological equipment with cell sorting options which enable an at least antiseptic operation. A sterile or even GMP-conforming process cannot be ensured and seems to be very difficult to realize also in the future. To ensure a high sample throughput, a rapidly flowing fluidic system is required. The shear forces occurring during the passage of the cells represent a massive impairment of cellular functions and clearly reduce viability in preparative applications and may result in long-lasting changes of the cellular morphology, growth and productivity or other specific functions. In addition, the selected cells are pooled; a safe collection of single cells does not appear to be realizable technically for these sample throughputs.
Cells can be cultured while embedded in viscous media (semisolid matrix). By a secretion assay, a secreted product can be stably precipitated in a stable cloud around the cell by means of a fluorescence-coupled detection system. This cloud, which exhibits a stronger fluorescence than that of the background can be utilized for a rough evaluation of high producers. In a second step, cells identified as high producers by an image analysis can be removed manually or automatically through a micromanipulator-operated glass capillary and cultured further (DE-A-10209788). However, this method has drawbacks relating to detection sensitivity and a limited sample throughput due to a low automatization potential.
The US-American company One-Cell Systems Inc. trades a special method for the FACS-supported sorting of gel microdrops by FACS. This ensures an at least cell-saving selection process, which is combined with the high sorting speed of FACS technology. Cells are included in gel microdrops having diameters of 20-100 μm. There, they secrete their product, which is bound to a special agarose matrix through a product-specific capture antibody. In a second step, the bound product is detected by a fluorescence-labeled product-specific antibody, and the number of cells is simultaneously determined due to the localization (FIG. 1b). The fluorescence signal can be used for sorting the drops by means of FACS technology. Subsequently, the matrix is dissolved again, and the cells are suspended.
In addition, there is also a possibility of isolating cells having a specific secretory activity by immunomagnetic separation (Miltenyi, Dynal Biotech, Polysciences). However, this method has the disadvantage that it does not allow for a quantification of the secretion performance and does not exhibit single cell specificity.
Therefore, there has still been a need for a method which can improve particular properties of a cell culture, such as the production performance, by a well-aimed selection of single cells and their subsequent further culturing in said cell culture.